Lantibodies were initially described by the inventors of this application (1). Lantibodies are derived from antibiotics which are a family of natural peptides that have antimicrobial activity. Lantibiotics have unique chemical and biological properties that are conferred by the presence of unusual amino acid residues such as dehydroalanine, dehydrobutyrine, lanthionine, and 3-methyllanthionine. The dehydro residues are electrophilic, and are capable of reacting with nucleophilic groups on polypeptide surfaces (1). By constructing suitable polypeptide environments around the dehydro residues, it is possible to control the reactivity and specificity of the dehydro residues, to react in a highly specific way with particular nucleophilic groups on the polypeptide surface. This reaction can alter the biological activity of the polypeptide surface, and if it is on a pathogen such as a bacterium or a virus, the activity of the pathogen can be destroyed. If the polypeptide surface is part of an enzyme, the activity of the enzyme can be altered in some useful way.
In this disclosure, a novel process by which the lantibodies are designed and constructed is described, and the lantibodies thus produced are more versatile in their use. Also disclosed is a novel means for screening the lantibodies to identify those which bind specifically to particular desired targets.
The inspiration for this invention is the mammalian immune system, in which stem cells differentiate into B-cells. This differentiation involves random recombination events among the variable regions of antibody genes, so that the resulting B-cell becomes programmed for the production of a particular antibody whose antigen-combining regions have been determined by a random process. The antibody that any B-cell can make is then displayed on the surface of the respective B-cell, and this surface antibody can interact with circulating pathogenic antigens. In the event that an antigen binds tightly to one of the displayed antibodies, the binding triggers cell division and further maturation of the B-cell into a plasma cell, which then produces and secretes large quantities of the antibody, which then leads to the destruction of the antigen (2–3).
Essential features of this natural process include the random generation of a population of antibodies, each of which is produced by a cell that displays the antibody that it is genetically programmed to make. Then, there is a highly-efficient process for the selection and amplification of those antibodies that bind to a specific antigen. The amplification is achieved by stimulating the division of those B-cells that display the antigen-binding antibodies.
Accordingly, a process is disclosed herein by which a population of bacterial cells are genetically programmed to produce a random population of lantibody molecules with each individual bacterial cell being dedicated to the production of a particular lantibody structure. Using discoveries in the Inventor's laboratory, the lantibody that a given bacterial cell produces is displayed on the surface of the cell. It is demonstrated that a population of lantibody producing cells can be exposed to an antigen, and that the cells whose surface lantibodies can bind to the antigen can be specifically recovered, so that the population of antigen-binding cells is enriched. This enriched population can be subjected to repeated selection and enrichment, so that a purified population of the specific antigen-binding cells can be obtained. Once the antigen-binding cells are obtained, the lantibody that is displayed on the surface of the cell can be determined by sequencing the genetic element that encodes the polypeptide sequence of the lantibody that the cell produces.
Knowledge of the structure of the lantibody can provide the basis of understanding the fundamental principles that are responsible for causing a particular lantibody to bind to a particular antigen. This knowledge can be applied to the rational design of new lantibodies that are directed toward nucleophilic targets, so that the methods by which new antibodies are made is not solely dependent on random chance.